Grapevines (Vitis) are a major global fruit crop with enormous economic and cultural significance, particularly Vitis vinifera, which is used for wine cultivation. A relatively small number of V. vinifera cultivars are used commercially to maintain consistency in the fruit as the plants are heterozygous and do not breed true. Thus, because so much of the commercial grape crop is dependent on these cultivars, which have limited diversity, disease resistance is of major concern. Classical breeding techniques to increase disease resistance generally erode fruit quality, making grapevines a prime candidate for genetic manipulation for improving disease resistance. However, the production of transgenic grapevines has proven difficult, as woody perennials, such as grapevines, are known to be recalcitrant to transformation, and the selection process required by Agrobacterium-mediated transformation is significantly more stringent due to out competition by the untransformed cells, leading to highly variable success rates (Mullins et al., Meth. Mol. Bio. 344:273-285, 1990); Bouquet et al., Methods Mol. Biol. 344:273-285, 2006). Thus, a need exists for reliable, efficient method for the delivery of genes to grapevines for disease treatments and the modification of grapevines for desired characteristics.
During the last two decades, viral vectors for the transient expression of the proteins in plants and animals became indispensable tools of molecular biology and biomedicine (Pogue et al., Annu. Rev. Phytopathol. 40: 45-74, 2002; Gleba et al., Curr Opin Biotechnol 18: 134-141, 1007). With the advent of RNA interference (RNAi) or RNA silencing, viral vectors were also developed for virus-induced gene silencing or VIGS (Godge et al., Plant Cell Rep 27(2):209-219, 2008; e-pub ahead of print, 2007). Taken together, an ability to rapidly overexpress or silence genes of interest made viral vectors important tools in functional genomics.
A number of plant viruses have been engineered into viral vectors, each with limitations and plant specificity. Most are suitable only for use in dicotyledonous herbaceous plants. By and large, icosahedral viruses are ill suited for accommodating foreign genes mostly due to the limited size of their capsids. In general, the elongated viruses have exhibited a better ability to tolerate recombinant genes and to express them to very high levels. Currently, the most commonly used vectors are those based on the rod-shaped Tobacco mosaic virus (TMV, genus Tobamovirus) (Pogue et al., Annu. Rev. Phytopathol. 40: 45-74, 2002; Gleba et al., Curr Opin Biotechnol 18: 134-141, 1007). These vectors are characterized by high expression levels but relatively low genetic stability, especially when it comes to large foreign inserts.
Another series of vectors is based on rod-shaped Tobacco rattle virus (TRV, genus Tobravirus) (Godge et al., Plant Cell Rep 27(2):209-219, 2008; e-pub ahead of print, 2007). Vectors derived from filamentous viruses are commonly based on Potato virus X (PVX, genus Potexvirus) (Chapman et al., Plant J 2: 549-557, 1992) and Tobacco etch virus (TEV, genus Potyvirus) (Dolja et al., Proc. Natl. Acad. Sci. USA 89: 10208-10212, 1992). TMV, TRV, and PVX vectors contain an expression cassette with a subgenomic RNA promoter, while TEV vectors use an alternative principle of protein expression based on a polyprotein processing. This latter feature provides the potyviral vectors with much higher genetic stability than that found in promoter-containing vectors (Dolja et al., Virology 252: 269-274, 1998).
Gene expression vectors based on Beet yellows virus (BYV, genus Closterovirus) have been developed (Hagiwara et al., J. Virol. 73: 7988-7993, 1999; Peremyslov et al., Proc. Natl. Acad. Sci. USA 96, 14771-14776, 1999). Although the levels of protein expression achievable for closteroviral vectors may be lower than those for TMV or TRV, these vectors have proved to be very stable genetically and capable of accommodating several expression cassettes based either on additional heterologous subgenomic RNA promoters or polyprotein processing. Such versatility of closteroviral vectors is most likely due to the large size of closteroviral genomes and presence of genes that dramatically increase genome replication and gene expression ability and possibly provide for increased fidelity of RNA copying (Dolja et al., Virus Res. 117: 38-51, 2006). Strong suppressors of RNAi (Reed et al., Virology 306: 203-209, 2003; Chiba et al., Virology 346: 7-14, 2006) and the leader proteinases of closteroviruses (Peng et al., J. Virol. 75(24), 12153-12160, 2001) are among the genes that ensure high genetic and evolutionary performance of closteroviruses and precondition their genomes for accommodating additional genes, viral or foreign.
One of the most critical characteristics of the viral vector is its host range that severely limits its potential utility for the desired crop plants. All of the vectors described above are able to infect only dicotyledonous herbaceous plants. In other words, the need to generate a viral vector for monocots or for woody crops such as grapevine dictates the need of using viruses that naturally infect such plants as a platform for vector development.
To date, very few viral vectors potentially suitable for woody plants have been developed, and data showing expression has typically been limited to a narrow range of model plants. One of these vectors is based on Apple latent spherical virus RNA 2 (ALSV, family Sequiviridae) (Li et al., Arch. Virol. 149: 1541-1558, 2004). Although the authors claim that ALSV vector was able to express the green fluorescent protein (GFP) by polyprotein processing upon mechanical inoculation to apple seedlings, no convincing experimental proof of such ability was presented in the paper. Similarly, no data is available to support recent claims of a ‘universal’ vector based on Tomato yellow leaf curl geminivirus, allegedly capable of systemically infecting a vast variety of plants from dicots to monocots to trees and vines (Peretz et al., Plant Physiol. 145(4):1251-1263, 2007). Another vector was developed using Grapevine virus A (GVA, a Vitivirus). Its ability to express a foreign protein was demonstrated in tobacco (Haviv et al., J. Virol. Meth. 132: 227-231, 2006) and remains unproven for grapevine. Another vector is based on Citrus tristeza virus (CTV), a closterovirus closely related to BYV (Folimonov et al., Virology 368(1):205-216, 2007). However, CTV is useful only in Citrus species, and its propagation involves cumbersome process of cycling in protoplasts prior to slash-inoculation of citrus trees with isolated virions. Accordingly, there exists a strong need for viral vectors suitable for transforming woody plants, particularly grapevines.